Taiwan was considered rabies-free for > 50 years until the disease reemerged in its ferret badger population (Melogale moschata) in 2013. Although it is still expanding slowly northward in eastern Taiwan, its spread in the west has thus far been halted by the Da-An River. To prevent diffusion of rabies, an immunization zone should be established along the epidemic boundary before further rabies expansion crosses it. This study developed 19 microsatellite markers with tri- or tetra-nucleotide motifs specifically for ferret badgers using next generation sequencing, and we used these markers to evaluate level of gene flow across the Da-An River. Low levels of genetic differentiation and high levels of gene flow across the river indicated that the Da-An River was not a permanent barrier to rabies. Rabies may eventually be able to expand across this temporary boundary. We suggest that an immunization zone should be established along the Da-An River as soon as possible.